Diagnostic performance and application of a real-time PCR assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal tract disease

ABSTRACT Johne’s disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis. As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

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Evaluation of the Diagnostic Performance of the XpertClostridium difficileAssay and Its Comparison With the Toxin A/B Enzyme-Linked Fluorescent Assay and In-House Real-Time PCR Assay Used for the Detection of ToxigenicC. difficile

Journal of Clinical Laboratory Analysis ◽

10.1002/jcla.21655 ◽

2014 ◽

Vol 28 (2) ◽

pp. 124-129 ◽

Cited By ~ 5

Author(s):

Dong Hee Whang ◽

Shin Young Joo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Diagnostic Performance ◽

Fluorescent Assay ◽

Pcr Assay ◽

Toxin A

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Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces

Parasitology ◽

10.1017/s0031182012000649 ◽

2012 ◽

Vol 139 (10) ◽

pp. 1266-1272 ◽

Cited By ~ 5

Author(s):

O. SANPOOL ◽

P. M. INTAPAN ◽

T. THANCHOMNANG ◽

P. SRI-AROON ◽

V. LULITANOND ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

High Sensitivity ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Intermediate Hosts ◽

Pcr Assay ◽

Fecal Samples ◽

Schistosoma Mekongi

SUMMARYSchistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

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